DE3900650A1 - VITAMIN B12 DETERMINATION - Google Patents

Abstract

To determine vitamin B12, a sample solution is incubated with at least two receptors R1 and R2, of which R1 mediates binding to the solid phase and R2 is labelled, where the receptor used as one of the two receptors R1 or R2 contains a monoclonal antibody which is capable of specific binding with B12 and has an affinity constant of at least 5 x 10<9> l/mol for B12, and the receptor used as the other receptor R2 or R1 contains B12 or an analogue thereof, and the two phases are separated and the labelling in one of the two phases is measured.

Description

Vitamin B12 or cobalamin is an essential vitamin found in body fluids such as whole blood, plasma, serum in low concentrations (about 10 ^-14 mol / l), for which in particular the strong binding to the B12 transport proteins (the trans-cobalamins) is remarkable , The manifestations of vitamin B12 deficiency that may be due to inadequate dietary vitamin intake, malabsorption syndromes, a genetically-induced deficiency of one or more trans-cobalamins, or the presence of intestinal parasites such as As the fish tapeworm (Diphyllobothria), can show in different appearances, which depend on the age of the individual and the duration of vitamin B12 insufficiency. A low vitamin B12 deficiency causes a reduction in red blood cells, which further causes a number of metabolic disorders, and megoblast anemia. In children, the nervous system is attacked, and in some cases, blindness can result.

The currently used methods for the determination of Cobalamines, especially of cyanocobalamin (vitamin B12) in very dilute aqueous solutions (such as blood serum) are based on methods using Radioactive tracers in which intrinsic factor (IF) is used as the binding reagent. The common techniques work using ⁵⁷Co-B12 as a marker on the basis of a competitive Principle in which free and labeled analytes um Compete the binding with the IF. The separation of  bound and free analyte (bound / free separation) then takes place due to methods such. B. using of activated carbon, solid phase IF or magnetic Separation, IF in paramagnetic particles is bound (see Brit J. Haemat 22 [1972], 21-31, Clin. Chem. 24 [1978], 460-466, Clin. Biochemistry 18 [1985], 261-266).

Before the determination of vitamin B12 in body fluids It is necessary to have vitamin B12 in his blood replace existing binding proteins. This is done, for example by heat or by destruction of the binding protein in the alkaline range (pH <13.5) under the action of the SH bond-splitting thiol Dithiothreitol (DTT) (Incubation of the serum sample by means of DTT in the alkaline range). By adding organic Fabrics, e.g. Acetone, or by adding competitive, cross-reacting species, e.g. As Cobinamide, this can Destruction be strengthened. Is advantageous in the Determination also alkali metal cyanide added to the Extend the ability to extract vitamin B12, and to make the cobalamins into a stable and detectable Form, namely the cyanocobalamin to convict.

The disadvantages of the known methods of determination of vitamin B12 are in particular in the use of Intrinsic factor justified. This will be wrong results observed when the intrinsic factor used is not is sufficiently pure (max 5% contamination by other B12 binding proteins). Numerous samples included apparently antibodies to IF, which the IF binding ability for radiolabeled B12. This can be too pretend low vitamin B12 levels.

Object of the present invention was therefore, a To provide a method for the determination of vitamin B12, with the on the use of intrinsic factor can be omitted and thus the above Disadvantages can be avoided, and that simple and reproducible way an accurate determination of vitamin B12 in serum.

The invention therefore relates to a method for Determination of vitamin B12 by incubation of a Sample solution with at least two receptors R₁ and R₂, of which R₁ mediates binding to the solid phase and R₂ is marked, separating the two phases and Measurement of the marking in one of the two phases, the characterized in that one of the both receptors R₁ or R₂ uses a receptor, one specifically bindable with B12, monoclonal Antibody that has an affinity constant of B12 for B12 has at least 5 × 10⁹ l / mol, contains and as others Receptor R₂ or R₁ uses a receptor, the B12 or an analogue thereof.

The inventive method means a crucial Advance for clinical diagnostics, as the Vitamin B12 determination was one of the last parameters for which no immunological test, using immobilized monoclonal antibodies commercially was available.

For the immunological method of determination according to the invention are in principle all common immunological Assays, such as radioimmunoassay, enzyme immunoassay, Fluorescence immunoassay, etc. Further, all are Process variants, such as competitive immunoassay, IEMA procedure, etc., applicable.  

To determine vitamin B12 has proven to be special suitably a competitive enzyme immunoassay or a Proved method according to the IEMA principle. In the competitive Enzyme immunoassay competes with the one to be determined B12 with a known amount of labeled B12 um the binding sites of the carrier-bound monoclonal Antibody. The test procedure can also be carried out this way be determined that the B12 to be determined and carrier-bound B12 around binding sites present in deficit of the labeled monoclonal antibody. The bound to the carrier-fixed B12 portion of the labeled monoclonal antibody is determined by the label certainly. This variant can also be modified be that the monoclonal antibody is not labeled is used. The to the carrier-fixed B12 bound antibody content is then determined by with a directed against the Fc portion of the antibody Antibodies incubated and the bound labeled portion is determined. In the IEMA process, marked, Monoclonal antibody added in excess. The excess labeled antibody not bound to B12 is using a hapten carrier matrix from the Solution removed. The different variants of this Test methods and details of how to do this Methods are described in detail in the literature. However, there are also other immunological haptens Determination method for B12 determination using the antibodies according to the invention are possible, as well as they z. B. in the German applications DE-P 38 34 766 or DE-P 38 22 750 are described.

According to the invention, at least one monoclonal antibody used specifically directed against vitamin B12 and an affinity constant of <5 × 10⁹ l / mol, preferably greater than 10¹⁰ l / mol and more preferably greater than 5 × 10¹⁰ l / mol and a cross-reactivity against  Methylcobalamin and cyanocobalamin of 100% against Cobinamide of <0.05%, against purinylcobinamide of 1.1%, against Cobryrinsäure diamide of <0.05% against 2-Hydroxy-5,6-dimethylbenzimidazolylcobamide of 1.5% and against (carboxy (2-cyanamino-4,5-dimethylphenyl) - amino) cobamid of 0.07%.

The monoclonal antibodies can be used as complete antibodies, chimeric antibodies or bivalent antibody fragments be used.

The test solution is used to determine vitamin B12 therefore with at least two receptors R₁ and R₂ incubated.

Receptor R₁ mediates the binding to the Solid phase. For this purpose, the receptor R₁ either directly or be bound to the solid phase via a spacer or in soluble form and only after Implementation of the immunological reaction immobilized become. Receptor R₁ either contains a specific with vitamin B12 bindable monoclonal antibodies or vitamin B12 or an analog thereof.

The binding to the carrier (immobilization) of the antibody or of B12 is carried out according to those skilled in the art Methods by adsorptive or chemical bonding or by binding through a specific binding pair. In this case, a partner of the binding pair immobilized while the other partner chemically B12 or the antibody is bound. About this binding pair can then the antibody or B12 before or immobilized during the immunological determination reaction become. Examples of such binding pairs are biotin-streptavidin / avidin, hapten-antibodies, Antigen-antibody, concavalin-antibody, sugar-lectin, Hapten-binding protein.  

As support materials for the immobilization of the invention Antibody or for the immobilization of B12 can be used materials such. B. tubes (tubes), microtiter plates, beads or microcarriers from plastics such as polystyrene, vinyl polymers, Polypropylene, polycarbonate, polysaccharides, silicones, Rubber, or even treated glass (cf., for example, E.T. Maggio, "Enzyme Immunoassay" CAC Press, Florida, 1980, in particular Pages 175 to 178; EP-A-063,064; Bioengineering 16 [1974], 997-1003; C.J. Sanderson and D.V. Wilson, Immunology 20 [1971], 1061-1065). In particular, as Support material coated with avidin or streptavidin Support material, in particular polystyrene, preferably prepared as described in EP-A 02 69 092, used.

Receptor R₂ likewise contains either vitamin B12 or an analogue thereof or a monoclonal antibody which is specifically bindable with vitamin B12 and is labeled. For labeling are the usual means for each determination method means. Thus radioisotopes, for example ⁵⁷Co, are used for labeling in a radioimmunoassay. For an enzyme immunoassay, all enzymes customarily used for this purpose, for example peroxidase or β- galactosidase, are suitable. For fluorescence immunoassay, the usual fluorescent groups are useful as markers. Details of these various test methods and method variants are known to the person skilled in the art. In an analogous manner as in the binding to the solid phase, the binding of the label to B12 or the antibody can be carried out via a specific binding pair.

The binding of the antibody or B12 to one of the abovementioned binding partner takes place after the Expert common methods, for example about Carbodiimide and hydroxysuccinimide.  

When labeling B12 with an enzyme becomes preferably a B12 conjugate of the formula (I)

wherein B12 is the radical formed from cyanocobalamin (vitamin B12) by cleavage of a -CONH₂ group and R is a linking moiety (spacer), and x is 0 or 1, and GP is the residue of a glycosyl-containing marker enzyme linked via a glycosyl radical the -NH-N = grouping is bound. In the formula (I), the -CONH moiety is preferably in the d position of the B12 moiety, and especially B12-d-CO-NH-N = Gp, and especially

B12-d-CO-NH-NH-CO-CH₂ (O-CH₂-CH₂) ₃O-CH₂-CO-NH-N = GP

used. As a labeling enzyme (GP) is preferably Peroxidase (POD) used.

The B12 conjugates of the formula (I) are the subject of German patent application P (Title: new Cobalamin acid hydrazides and cobalamin derived therefrom Conjugates) of the same applicant with the same Filing. They can be controlled by coupling (condensation) the cobalamin acid hydrazides of the formula

(in which B12, R and x have the abovementioned meaning), which are likewise the subject of the aforementioned simultaneously filed German patent application P, with the OH groups of glycosyl residues of the glycoproteins after their oxidation and formation of the hydrazone moiety -NH-N = CH- Produce glycoprotein under conditions known per se.

In a preferred embodiment of the invention Procedure, the sample solution for replacement of vitamin B12 from the binding proteins in conventional Be prepared by adding the binding proteins by adding an SH group-cleaving thiol, dithiothreitol (DTT) in the alkaline range (pH <13.5), or else by boiling for 30-60 minutes and then Centrifugation be destroyed.

Preferably, in the method according to the invention for vitamin B12 determination the sample preparation (Cleavage of the binding protein) with lipoic acid (Liponic acid, LA) or a homologue thereof of the formula (II)

wherein n is 1 to 8, and especially 3 to 5, wherein the lipoic acid (formula II, n = 4) is particularly preferred.

This procedure for sample preparation is the subject German patent application P (title: Method for detaching an analyte from its bandage protein) of the same applicant with the same Filing. After this procedure, the incubation the sample at room temperature in the alkaline range (pH 10 to 14, preferably using Sodium hydroxide as alkaline medium, with a Concentration of 0.05 to 1 mmol / L) in less than 15 Minutes.

The acid of the formula (II) is used here (calculated for lipoic acid with n = 4), preferably in a range of 1 to 20 mg / ml, and in particular in the range of 4 to 10 mg / ml.

The inventive method provides very accurate and reproducible values, which is mainly due to this is that a monoclonal antibody to vitamin B12 is used, which has a very high affinity cons has constant resistance to vitamin B12. These antibodies are also the subject of the invention. Such spe cif monoclonal antibodies of such high Affinity constants are not yet known.

Another object of the invention is a method for producing a B12-specifically bindable, monoclonal antibody characterized is that one inbred mice with vitamin B12-d-acid, to which via a spacer, in particular 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide is an immunogenic carrier material is coupled, immunized, B lymphocytes of the immunized animals wins and transforms Agents fused with myeloma cells so formed Hybrid cells are cloned and cultured and the monoclonal Antibody isolated from these cells.

To obtain the monoclonal anti B12 initially undergoes an immunogenic carrier material linked. As immunogenic carrier materials All are commonly used for this purpose substances, such as albumins, such as bovine serum, Albumin, edestin, etc. Linking B12 with the Support material is carried out by methods known per se.

Subsequently, experimental animals, for example Mice immunized with the immunogenic conjugate. to Immunization is the immunogen, for example in Kom administration with the adjuvant in the usual way. Preference is given as adjuvant complete or incomplete tes Freunds adjuvant used. The immunization is preferably carried out over several months with at least four immunizations four to six weeks apart (Injection intraperitoneally).  

From thus immunized animals B lymphocytes are won which fuses with a permanent myeloma cell line become. The fusion takes place according to the known one Method of Köhler and Milstein (Nature 256, 1975, Pages 495 to 497). The hereby formed Primärkul Structures of hybrid cells are in the usual way, for. B. using a commercial Zellorters or by "limiting dilution", cloned. There will be each the crops processed in a suitable Test method, for example, an enzyme immunoassay (ELISA method), are positive against B12 and the show cross-reactivity mentioned. You get like that several hybridoma cell lines containing the inventive produce monoclonal antibody. After known Methods can cultured these cell lines and those of they isolated monoclonal antibodies isolated become.

In this way, the inventively used Antibodies are obtained, in particular antibodies with an affinity constant of <5 x 10⁹ l / mol, preferably greater than 10¹⁰ l / mol, more preferably greater than 5 × 10¹⁰ l / mol and a cross-reactivity against methylcobalamin and Cyanocobalamin of 100% against cobinamide of <0.05%, against Purinylcobinamide of 1.1%, against Cobryrinsäure diamide of <0.05%, against 2-hydroxy-5,6-dimethylbenzimide azolylcobamide of 1.5% and against (carboxy (2-cyanamino) 4,5-dimethylphenyl) amino) cobamide of 0.07%. Antibodies that show such high specificity z. B. from the thus obtained cell lines ECACC 88101301 and ECACC 88101302.

The cell lines are below the given number at the depository ECACC (European Collection of Animal Cell Cultures, Proton Down, UK).  

The monoclonal antibodies thus obtained are characterized by the fact that they have a very high affinity (affinity constant greater than 5 × 10 ^-9 ) for B12 and the mentioned cross-reactivities. Preferably, the affinity of the monoclonal antibodies is above 10¹⁰ l / mol, more preferably above 5 × 10¹⁰ l / mol.

The monoclonal antibodies of the invention are suitable excellently, in a sample, for example Serum or plasma to specifically determine the B12. For These determination methods can be monoclonal Antibodies as such are chimeric antibodies or fragments of which, the corresponding immunological Have properties, for example color fragments, be used. Under the term "monoclonal anti Therefore, both full antibodies and also understood the fragments.

The following examples are intended to illustrate the invention in more detail explain without limiting it. Under space Temperature (RT) becomes a temperature of 25 ° C ± 2 ° C Roger that. Percentages are by weight percent.

Fig. 1 shows a standard curve for a vitamin B12 determination according to Example 4 with various concentrations which MAK:

Curve 1: 85 ng / ml MAK,
Curve 2: 90 ng / ml MAK,
Curve 3: 95 ng / ml MAK,
Curve 4: 100 ng / ml MAK.

Fig. 2 shows a comparison of a determination according to Example 4 (curve 2) with a determination using polyclonal antibodies (curve 1).

Example 1 Obtaining Monoclonal Antibodies to Vitamin B12 Preparation of the immunogen

Vitamin B12-d-acid (manufactured according to JACS 102 (1980) 2215) is converted via 1-ethyl-3- (3-dimethylaminopropyl) car bodiimide (EDC) coupled to edestin.

Immunization of mice with vitamin B12 conjugate

Balb / c mice, 8 to 12 weeks old, were given 100 μg Immunogen in complete Freunds adjuvant intraperi tonal first. Three weeks later, after six weeks further immunizations at four-week intervals carried out. In this case, 100 ug immunogen in inc administer Freund's adjuvant intraperitoneally enough. 4 days, 3 days and 2 days before the merger was immunized again with 100 μg immunogen in vitro.

fusion

Spleen cells of an immunized mouse were used with P3 × 63Ag8-653 myeloma cells (ATCC-CRL 8375) in proportion Mixed 1: 5 and centrifuged (10 minutes, 300 g, 4 ° C). The cells were resuspended with BSS (Balanced Salt Solution) buffer and 400 g in one Centrifuge 50 ml tip tubes. The supernatant was dumped, the cell pellet fanned, 1 ml of PEG (MG 4000, Merck) added and pipetted. To one minute in a water bath were at room temperature 5 ml RPMI 1640 medium (RPMI = Rosewell Parker Memory Institute) without FCS (fetal calf serum) at a time dropped from 4 to 5 minutes, mixed, on  50 ml filled with medium and then 10 minutes centrifuged at 400 g, 4 ° C. The sedimented cells were taken up in RPMI 1640 medium + 10% FCS and 5 x 10⁴ to 1 x 10⁵ spleen cells or 5 x 10⁴ peritoneal exu dat cells added as "feed cells". The next day hypoxanthine-azaserine selection medium (100 mmol / l Hypoxanthine, 1 μg / ml azaserine).

About 7 to 10 days after the merger were already many Clones visible. The supernatant of the primary cultures was according to an ELISA method described in Example 2 tested. Primary cultures containing the desired cross were shown by FACS (Fluorescence activated cell sorter) in 96-well cell culture plates cloned. As "food cells" were 1 × 10⁴ peritoneal exu dat cells or 2x10⁴ spleen cells per well. In this way, for example, the two could Hybridoma cell lines

ECACC 88101301 and
ECACC 88101302

be deposited with the depositary ECACC under the respectively specified deposit number have been submitted.

Induction of ascites

5 × 10⁶ hybrid cells were i. p. in once or twice with 0.5 ml of pristane pre-treated mice injected. ascites with an IgG concentration of 5 to 20 mg / ml, 1 be won until 3 weeks later. This can be in usually the antibodies are isolated. These Monoclonal antibodies are specific to vitamin B12 directed and show the desired cross-reactivity. The monoclonal antibodies are probed with MAK 1 (from ECACC 88101301) or MAK 2 (from ECACC 88101302).  

example 2 Screening test for antibodies to vitamin B12

In order to know the presence and specificity of antibodies against vitamin B12 in the serum of immunized mice or in the culture supernatant of the hybrid cells or in ascites, an ELISA method is used as test principle: microtiter plates are incubated with 1 μg / ml B12 conjugate (B12 acid-coupled to bovine serum albumin over EDC) coating buffer (0.2 M sodium carbonate / bicarbonate, pH 9.3 to 9.5) at 37 ° C for one hour. It is aftertreated for 10 minutes with 0.9% sodium chloride solution and 1% albumin solution. At closing is washed with 0.9% sodium chloride solution ge. Thereafter, incubated at 37 ° C for one hour with 100 ul of sample and washed again with 0.9% Natrumchloridlö solution. To test the cross-reaction, 50, 500 and 5000 μg / ml of the vitamin B12 derivative to be tested are added to the sample solution. A reduction of the measurement signal in the presence of the derivatives means cross reaction. Another incubation (one hour, 37 ° C) with 450 U / ml of a sheep Fab anti-mouse Fcγ-peroxidase conjugate follows. After a new washing step with 0.9% sodium chloride, the Peroxidaseak activity is determined in a conventional manner (for example, with 2,2'-azino-di- [3-ethylbenzthiazolin-sulfonate (6)] (ABTS®), 30 minutes at room temperature , read the Ex inkktionsdifferenz, Δ mE , at 422 nm).

example 3 Determination of the cross reaction

The test is, as described in Example 2, Runaway leads.

The monoclonal antibody is in each case on Cross-reaction to antigen to be tested in increasing concentrations tration (50 μg, 500 μg, 5000 μg / ml). The Cross reaction is then according to the following formula calculated:

C = Concentration of the antigen to reach 50% of the max. Signal is required.

The following table shows the determined values, the same for the monoclonal antibodies MAK 1 and MAK 2 are, put together.

cross-reacting antigen cross reaction Methyl-cobalamin 100 Cyano-cobalamin 1000 cobinamide <0.05 Purinylcobinamid 1.1 Cobryinsäure diamide <0.05 2-hydroxy-5,6-dimethyl-benzimidazolylcobamid 1.5 (Carboxy (2-cyanamino-4,5-dimethylphenyl) aminocobamid 0.07

example 4 Determination of vitamin B12 a) Sample preparation

250 μL of human serum is mixed with 125 μL of the separation reagent (consisting of 8 mg / ml lipoic acid, 1 mg / ml potassium cyanide dissolved in 0.5 mol / l NaOH) and 15 Incubated for a few minutes at room temperature. Subsequently 125 μl, 200 mmol / l phosphate buffer, pH 4.1 added.

b) Reagents

Polystyrene tubes coated with Thermo-RSA Streptavidin (prepared according to EP-A 02 69 092).

reagent 1

95 ng / ml biotinylated MAK 1 or MAK 2 (Biotiny lation according to JACS 100 (1978) 3585 to 3590).

reagent 2

B12-d-CO-NH-NH-CO-CH₂ - (- O-CH₂-CH₂-) ₃-O-CH₂-CO-NH-N = POD) (activity about 60 mU / ml).
40 mmol / l phosphate buffer pH 7.2.

reagent 3

100 mmol / l phosphate citrate buffer, pH 4.4
1.9 mmol / l ABTS®,
3.2 mmol / l sodium perborate.

c) implementation of the provision

To carry out the determination, 200 .mu.l ago treated sample with 800 μl reagent 1 in Given streptavidin-tube and added for 60 minutes Room temperature incubated. Subsequently, with Washed washing solution and 1000 .mu.l of reagent 2 added and incubate for 30 minutes at room temperature. It is washed with washing solution and 1000 .mu.l Reagent 3 added, for 30 minutes at room temp Incubation and the formed color as a measure measured for the vitamin B12 content at 422 nm.

d) Analogous results are obtained if instead of biotinylated from biotinylated complete MAK 1 Fab fragments are used. Fab fragments are made as follows:

With MAK 1, as in Biochem. J. 73 (1959) 119 to 126, performed a papain splitting. The resulting Fab fragments are by means of Gel filtration over Sephadex G 100 and Ionenaus exchange chromatography on DEAE cellulose Meth. In Enzymology 73 (1981) 418 to 459 abge separates.

example 5 Comparison with a known radioimmunoassay for B12

As a standard, cyanocobalamin in 40 mmol / l Phos phosphate buffer, pH 7.2 with 0.9% sodium chloride, 0.9% Crotein C and 0.1% potassium cyanide used. To Ver the same was the test sold by Becton Dickinson  (Simultaneous No Boil SNB-B12 / folate radioassay) ver applies. In this test, immobilized intrinsic Factor and radioactively labeled B12 (⁵⁷Co B12) use det. For sample preparation Dithio Threitol (DTT) used in alkaline solution. The Correlation between this radioimmunoassay and the inventive method is in the field of a Vita min-B12 concentration between 100 and 1400 pg / ml <0.98.

example 6 Vitamin B12 determination with polyclonal antibody against B12 (comparative example) a) Obtaining the antiserum

10 sheep are described in Example 1 immunogen (0.5 ng / ml in complete Freund's Adjuvant) at four-week intervals over 6 months immunized. Subsequently, the antiserum recovered and purified by affinity chromatography.

b) Preparation of biotinylated Fab fragments of polyclonal antibody against B12 (Fab biotin

With the polyclonal antibodies, as in Biochem. J. 73 (1959) 119-126, a Papain splitting performed. The entstan which fragments are transformed by gel filtration Sephadex G100 and Ion Exchange Chromatography on DEAE-cellulose after Meth. in Enzymology 73 (1981) 418-459 separated. The biotinylation is carried out as described in JACS 100 (1978) 3585-3590.  

c) implementation of the provision

The execution of the determination takes place, as in Example 4, wherein instead of 95 ng / ml biotinylated MAK 1 95 ng / ml Fab-biotin be used.

Fig. 2 shows the comparison between a B12 Be mood over polyclonal and monoclonal antibodies. It then turns out that a much steeper calibration curve is obtained with the monoclonal antibodies according to the invention.

Claims (16)

1. A method for the determination of vitamin B12 by incubation of a sample solution having at least two receptors R₁ and R₂, of which R₁ imparts the binding to the solid phase and R₂ is marked, separation of the two phases and measurement of Mar kierung in one of the two phases, characterized in that one uses as one of the two receptors R₁ or R₂ a receptor containing a specifically bindable with B12, monoclonal antibody having an affinity constant of at least 5 × 10⁹ l / mol for B12, and as another recipe R₂ or R₂ R₁ uses a receptor containing B12 or an analogue thereof. 2. The method according to claim 1, characterized, that one is capable of binding with B12 monoclonal antibodies use an antibody det, which has a cross-reactivity to methylcobal min and cyanocobalamin of 100%; against cobinamide of 0.05%; against purinylcobinamide of 1.1%; against cobryric acid diamide of 0.05%; against 2-hydroxy-5,6-dimethylbenzimidazolyl cobamide of 1.5% and against (carboxy (2-cyanamino-4,5-dimethyl phenyl) -amino) -cobamide of 0.07%. 3. The method according to claim 1 or 2, characterized, that as a monoclonal antibody one of the Cell line ECACC 88101301 or ECACC 88101302 pro used reduced antibody.   4. The method according to any one of the preceding claims, characterized, that as a receptor R₁ a conjugate of biotin and a monoclonal B12 capable of binding specifically with B12 used as the solid phase a streptavidin-coated carrier material is used. 5. The method according to any one of the preceding claims, characterized in that as receptor R₂ a B12 conjugate of the following formula: wherein B12 is the radical formed from cyanocobalamin by cleavage of a CONH₂ group and R is a spacer, x is 0 or 1, and GP is the residue of a glycosyl-containing marker enzyme linked to the NH-N = moiety via a glycosyl radical , 6. The method according to claim 5, characterized characterized in that as B12 conjugate B12-d-CO-NH-NH-CO-CH₂ (O-CH₂-CH₂) ₃ -O-CH₂-CO-NH-N = GP used, wherein GP is the peroxy that means first. 7. The method according to any one of the preceding claims, characterized in that the sample solution before incubation with the two receptors R₁ and R₂ by adding an acid of the general formula (I): pretreated in which n is an integer from 1 to 8. 8. The method according to claim 7, characterized characterized in that one Pretreatment lipoic acid used. 9. The method according to any one of the preceding claims, characterized, that a monoclonal antibody is used, its affinity constant for B12 is greater than 10¹⁰ l / mol. 10. The method according to claim 9, characterized characterized in that a mono clonal antibody is used, whose affin constant for B12 greater than 5 × 10¹⁰ l / mol. 11. Method of Making One Specific to B12 bindable, monoclonal antibody, characterized, that inbred mice with vitamin B12-d-acid, to which via a spacer, in particular 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide an immunogenic Carrier material is coupled, immunized, B-lympho cyten of the immunized animals wins and trans forming agents fused with myeloma cells, the hybrid cells thus formed are cloned and cultivated fourth and the monoclonal antibody from these Isolated cells.   12. Cell line ECACC 88101301. 13. Cell line ECACC 88101302. 14. A reagent for the determination of B12, containing one the binding to the solid phase mediating recipe tor R₁ and a labeled receptor R₂, characterized, that it is one of the two receptors R₁ or R₂ a receptor that is specific to B12 bindable monoclonal antibody suitable for B12 an affinity constant of at least 5 x 10⁹ l / mol contains, and as another receptor R₂ or R₁ contains a receptor, the B12 or an analogue gon thereof and, where appropriate, an after white system for marking. 15. A reagent according to claim 14, characterized in that it as a solid phase coated with avidin or streptavidin matrix, as a receptor R₁ a conjugate of biotin and a B12 specifically bindefähi conditions, monoclonal antibody, as R₂ a connec tion of the Fomel B12- CO-NH (NH-R-NH) _x N = GP, where B12 is the residue formed from cyanocobalamin by cleavage of a CONH₂ group, R is a spacer, x is 0 or 1 and GP is the residue of a glycosyl-containing marker enzyme which has a Glykosylrest is bound to the NH-N = moiety and contains a suitable for the marker enzyme detection system. 16. A reagent according to any one of claims 15 or 16, characterized in that it additionally comprises a compound of formula wherein n is 1 to 8 contains.